Functional cooperation of the glycine synthase-reductase and Wood–Ljungdahl pathways for autotrophic growth of Clostridium drakei

Abstract

Among CO 2 -fixing metabolic pathways in nature, the linear Wood-Ljungdahl pathway (WLP) in phylogenetically diverse acetateforming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO 2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genomescale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13 C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO 2 , subsequently converting CO 2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO 2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.CO 2 fixation | acetogen | Wood-Ljungdahl pathway | systems biology | glycine synthase-reductase pathway T he linear Wood-Ljungdahl pathway (WLP) in anaerobic acetogens is considered the most energetically efficient pathway to convert CO 2 to formate and then into acetyl-CoA. With this advantage, acetogens are considered to be the most promising industrial platform to produce biofuels and chemical commodities through synthesis gas fermentation (1-4). Although gene composition and arrangement of the WLP vary among acetogens, the WLP-coding genes are well-conserved, along with two genes encoding a partial glycine synthase: the glycine cleavage system H protein (gcvH) and dihydrolipoyl dehydrogenase (lpdA) genes (5-8). The glycine synthase pathway was initially proposed for the utilization of CO 2 under autotrophic growth conditions (2). While the two genes are well-conserved in the gene cluster, other genes in the glycine synthase pathway are missing in many acetogen genomes, which raises questions regarding a potential functional role of these enzymes under autotrophic growth conditions. Following synthesis, glycine can be reduced to acetyl-phosphate (acetyl-P), which is likely to be converted into acetate by acetate kinase (ackA), thereby producing one ATP, termed the glycine synthasereductase pathway (GSRP). Alternatively, serine hydroxymethyltransferase (SHMT) converts the produced glycine to serine, which then becomes transformed to pyruvate and biomass (9-11). Recently, an artificial metabolic pathway constructed with glycine synthase and SHMT, termed the reductive glycine pathway (RGP), has shown the capability of fixing CO 2 using alternative electron donors (12,13). Despite sharing common reactions and the presence of genes encoding a partial glycine synthase, the functional role of the pathway in the presence of intact WLP has remained uncertain.In this study, we elucidated the role of the GSRP a...

The reductive glycine pathway allows autotrophic growth of Desulfovibrio desulfuricans

“…As the reductive acetyl-CoA pathway is not complete in D. desulfuricans, the rGly pathway is the sole carbon fixation pathway in this bacterium. In summary, our and other recent findings suggest that the rGly pathway is phylogenetically widespread allowing autotrophic growth in Gram-negative bacteria as shown here for D. desulfuricans, and in Gram-positive bacteria, such as anaerobic clostridia (in cooperation with the reductive acetyl-CoA pathway) 33,34 . Most of the enzymatic components of the rGly pathway are present in many microorganisms 29 .…”

“…As "Candidatus Phosphitivorax anaerolimi" has yet to be isolated and no physiological evidence was provided for the activity of the suggested route, it remains unclear if this bacterium indeed grows autotrophically and uses the rGly pathway. Very recently, it was observed that the acetogen Clostridium drakei combines the GR variant of the rGly pathway with the reductive acetyl-CoA pathway during autotrophic growth on H 2 /CO 2 in the presence of yeast extract 34 . As the reductive acetyl-CoA pathway is not complete in D. desulfuricans, the rGly pathway is the sole carbon fixation pathway in this bacterium.…”

Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway

“…In various species of aerobic and anaerobic bacteria, as well as in plants and animals, GCS acts in the physiological direction of glycine decomposition. Recently, a biosynthetic role for the system acting in the opposite direction of glycine synthesis in cooperation with the WLP was proposed in the acetogen C. drakei under autotrophic conditions (104). A clear demonstration that the GCS functions in the direction of glycine synthesis for biomass assimilation from C 1 substrates was provided from metabolic engineering studies (105) in which unequivocal 13 C isotopic labeling experiments proved that the GCS functions as a central module in an engineered strain of E. coli capable of using CO 2 and either formate or methanol as the sole source of carbon and energy for growth.…”

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

“…The vectors contain both pIP404 and ColE1 of gram-positive and gram-negative bacteria origin, respectively. The antibiotic resistance gene is catP , which encodes for chloramphenicol acetyltransferase; hence, chloramphenicol and thiamphenicol can be used as selection markers [ 44 , 50 , 51 , 52 , 53 ]. In addition to these two plasmids, the pMTL80000 modular vector series was developed to transfer foreign DNA between Clostridium species and E. coli .…”

“…Among the acetogens, Clostridium drakei was found to have the GSRP, and it was demonstrated that the methyl branch of the WL pathway and GSRP can be used together to reduce CO 2 . According to these results, when the genes of gcvTH, gcvPA/B, grdX, trxAB , and grdABCDE constituting GSRP were introduced into E. limosum , which is known to lack GSRP, the growth rate and CO 2 consumption rate of the GSRP-introduced strain increased 1.4-fold, and acetate production increased 2.1-fold compared to the wild-type strain ( Figure 2 and Table 3 ) [ 52 ]. This result expanded the pioneering perspective for increasing the efficiency of C1 gas utilization by introducing a pathway to cooperate with the WL pathway in acetogens.…”